Kinetics and temperature dependence of exposure of endocytosed material to proteolytic enzymes and low pH: Evidence for a maturation model for the formation of lysosomes

Abstract

The temperature dependence of acidification of internalized dextran by Swiss 3T3 cells was determined using dual fluorescence flow cytometry. Essentially no acidification was observed at 11 degrees C; acidification was limited to pH 6-6.5 at temperatures between 13 degrees C and 17 degrees C. In contrast, a rapid drop to pH 6-6.5 followed by acidification to pH 5-5.5 was observed at temperatures above 19 degrees C. These results confirm the biphasic nature of the acidification process (J. Cell Biol. (1984) 98: 1757-1762). The timing of exposure of material internalized by fluid-phase endocytosis to lysosomal enzymes was determined for Swiss 3T3 cells by using a fluorogenic substrate specific for Cathepsin B. Hydrolysis of the substrate, as measured by both fluorometry and flow cytometry, began within minutes of its addition to cells at 37 degrees C, and was inhibited by coincubation with leupeptin, a competitive inhibitor of the enzyme, or by weak bases, which raise the pH of acidic compartments. At temperatures between 13 degrees (and 21 degrees C, the rate of hydrolysis was reduced to 31-44% of that at 37 degrees C. Thus, in contrast to previous reports, exposure of endocytosed material to at least one lysosomal enzyme is not inhibited below 20 degrees C; the reduction in hydrolysis rate may be explained by the temperature effects on the efficiency of the enzyme. The results for acidification and proteolysis are consistent with, but do not prove, a maturation model for the formation of lysosomes. We suggest that at lower temperatures, part of the maturation involving recycling and/or concentration of the contents of the endosome is inhibited. This causes the endosome to remain as a mildly acidic, low-density organelle containing lysosomal enzymes.

Acidification of morphologically distinct endosomes in mutant and wild-type Chinese hamster ovary cells.

“…Endosomes of the CHO mutants DTG 1-5-4 and DTF 1-5-1 (13, 27, 28) also show differences in pH, along with a marked decrease in early acidification (40). These and other studies on the kinetics of endosome acidification (12,29,32) show that endosomes within the same cell can vary in their pH.…”

“…Endosomes of the CHO mutants DTG 1-5-4 and DTF 1-5-1 (13, 27, 28) also show differences in pH, along with a marked decrease in early acidification (40). These and other studies on the kinetics of endosome acidification (12,29,32) show that endosomes within the same cell can vary in their pH.Differences in acidification may be important in determining the function of an endocytic compartment, because endocytic processes vary in their acidification requirements. Low density lipoprotein (LDL) and ct2M need only mildly acidic conditions (pH 6.5-6.7) to dissociate from their receptors (1, 17), whereas lysosomal enzyme dissociation (8), iron release (3,15,26), and penetration of diphtheria toxin (5, 31) all require a pH of <6.0.…”

“…Low density lipoprotein (LDL) and ct2M need only mildly acidic conditions (pH 6.5-6.7) to dissociate from their receptors (1, 17), whereas lysosomal enzyme dissociation (8), iron release (3,15,26), and penetration of diphtheria toxin (5, 31) all require a pH of <6.0. Therefore, measuring the pH of a specific type of endosome may help elucidate the role that endocytic compartment has in a particular physiologic process.From kinetic studies (12,29,32,40) it is not clear whether 9 The Rockefeller University Press, 0021-9525/87/12/2723/11 $2.00 …”

“…From kinetic studies (12,29,32,40) it is not clear whether 9 The Rockefeller University Press, 0021-9525/87/12/2723/11 $2.00…”

“…From kinetic studies (12,29,32,40) it is not clear whether progressive acidification results from pH changes within an organelle or from passage among organelles that are regulated to different pH values. Several different types of endosomes have been described in CHO cells (34,39,41).…”